Drug resistance has emerged as a major problem in the treatment of all microbial agents and in cancer chemotherapy. Drug resistance has become particularly acute in malaria where resistance to chloroquine, the cheapest and most efficacious antimalarial has spread throughout the endemic parts of the world and resistance to other antimalarials is rapidly developing and spreading. The goal of this work is to understand the mechanism of drug resistance and to eventually use that information to develop new approaches to chemotherapy. Discoveries over the last decade have identified several classes of ATP dependent transporters which often have broad substrate specificity and many of these have now been associated with conferring drug resistance to a broad range of xenobiotic compounds. The focus of our work has been on the role of these ABC (ATP-binding cassette) transporters in drug resistance in malaria. In previous work funded under this grant we have identified two ABC-transporters, PfMDR1 and PfMDR2 and evaluated the role of these genes in drug resistance. The approach we have taken is a functional analysis of the role of these genes in drug transport and resistance through gene identification and characterization, gene expression analysis, protein analysis and expression in homologous and heterologous systems. In parallel we have begun a similar analysis in Leishmania as a model system for ABC-transporter mediated resistance in protozoan parasites. Work in that system has led us to propose a new model for drug resistance in which drugs are sequestered by transport into specific subcellular compartments rather than directly effluxed from the plasma membrane as is the case in mammalian multidrug resistance mediated by ABC-transporters. This combination of approaches has proven valuable in the analysis of drug resistance in other microbial systems and in multidrug resistance neoplastic cells and has proven equally valuable in demonstrating the role of certain members of this ABC transport family in drug resistance in protozoan parasites. Our results clearly demonstrate both the presence of ABC-transporters and the role of at least two of these in drug resistance in protozoan parasites. The current proposal extends this work to include both characterization at the molecular level the mechanism by which these ABC-transporters modulate drug resistance and identification of additional ABC-transporters with particular emphasis on the PDR5/SNQ2 and MRP-related molecules.